Mutation of the BRAF oncogene is a common event in colorectal cancer (CRC). These mutations are associated with adverse outcome and insensitivity to epidermal growth factor receptor (EGFR) based therapy. Whereas RAF inhibitors have been successful in the treatment of BRAF-mutant malignant melanoma, response rates in BRAF-mutant CRC are surprisingly low. The basis for these disparate treatment outcomes remains incompletely understood. Preliminary studies from our groups found that suppression of the MAPK pathway by PLX4720 is incomplete in CRC lines. In some cases, this may be due to augmented EGFR-dependent signaling, but this does not explain all resistance in this setting. Our objective is to identify mechanisms operant in BRAF-mutant colorectal cancer that confer de novo resistance to RAF inhibitors, in hopes of enabling more efficacious therapeutics. First, differentially expressed genes linked to BRAF-mutant CRC will be identified by analysis of the TCGA dataset; these genes will be integrated with those that modify response to MAPK pathway inhibitors based on ongoing systematic functional screens. Top-ranking genes will be subjected to mechanistic studies to elucidate the molecular basis by which they confer resistance, in parallel, ongoing functional screens will be expanded to identify genes that are synthetic lethal with RAF inhibition in BRAF-mutant colorectal cells. Here, validation of leading candidates will be prioritized for genes that are potentially druggable-several candidates have already been nominated. Rational combinations of targeted agents with RAF inhibitors will be explored in cell culture and in xenograft models. Finally, clinical trials of combined RAF and MEK inhibitors will be performed at DF/HCC in an attempt to improve efficacy by enhancing suppression of the MAPK pathway and possibly prevent the emergence of drug-resistance. Tumor biopsies will be collected pre-treatment, on-treatment and post-progression, and whole exome and transcriptome sequencing will be used to identify genomic alterations that may drive resistance. Altogether, this work should provide a rigorous analysis of resistance to MAPK inhibitors and new therapeutic approaches to overcome them.